Interleukin 1 beta and Matrix Metallopeptidase 3 Contribute to Development of Epidermal Growth Factor Receptor-Dependent Serrated Polyps in Mouse Cecum.

BACKGROUND & AIMS: Transgenic mice (HBUS) that express the epidermal growth factor receptor (EGFR) ligand HBEGF (heparin-binding epidermal growth factor-like growth factor) and a constitutively active G protein-coupled receptor (US28) in intestinal epithelial cells develop serrated polyps in the cecum. Development of serrated polyps depends on the composition of the gut microbiota and is associated with bacterial invasion of the lamina propria, accompanied by induction of inflammation and up-regulation of interleukin 1 beta (IL1B) and matrix metalloproteinase (MMP) 3 in the cecum. We investigated the mechanisms by which these changes contribute to development of serrated polyps. METHODS: We performed studies with C57BL/6 (control) and HBUS mice. To accelerate polyp development, we increased the exposure of the bacteria to the lamina propria by injecting HBUS mice with diphtheria toxin, which binds transgenic HBEGF expressed by the epithelial cells and causes apoptosis. Mice were given injections of IL1B-neutralizing antibody and the MMP inhibitor N-isobutyl-N-(4-methoxyphenylsulfonyl)glycyl hydroxamic acid. Intestinal tissues were collected from mice and analyzed by histology, reverse-transcription polymerase chain reaction, enzyme-linked immunosorbent assay, immunofluorescence, and flow cytometry. We examined fibroblast subsets in polyps using single-cell RNA sequencing. RESULTS: Administration of diphtheria toxin to HBUS mice accelerated development of serrated polyps (95% of treated mice developed polyps before 100 days of age, compared with 53% given vehicle). IL1B stimulated subsets of platelet-derived growth factor receptor alpha+ (PDGRFA+) fibroblasts isolated from cecum, resulting in increased expression of MMP3. Neutralizing antibodies against IL1B or administration of the MMP inhibitor reduced the number of serrated polyps that formed in the HBUS mice. Single-cell RNA sequencing analysis showed subsets of fibroblasts in serrated polyps that express genes that regulate matrix fibroblasts and inflammation. CONCLUSIONS: In studies of mice, we found that barrier breakdown and expression of inflammatory factors contribute to development of serrated polyps. Subsets of cecal PDGFRA+ fibroblasts are activated by release of IL1B from myeloid cells during the early stages of serrated polyp development. MMP3 produced by PDGFRA+ fibroblasts is important for serrated polyp development. Our findings confirm the functions of previously identified serrated polyp-associated molecules and indicate roles for immune and stromal cells in serrated polyp development.

Mitochondrial pathways are involved in Eimeria tenella-induced apoptosis of chick embryo cecal epithelial cells.

Accumulating evidence suggests that Eimeria tenella severely damages the intestinal mucosa in infected poultry, resulting in deadly haemorrhagic typhlocolitis and major economic losses. Damage to host tissue is believed to arise mainly from apoptosis, which is, in general, intimately related to mitochondrial function. However, it is unclear whether mitochondria-dependent apoptotic pathways are specifically involved in parasite-induced apoptosis of chick embryo cecal epithelial cells. Because the mitochondrial permeability transition pore (MPTP) and caspase-9 are important elements in these pathways, we studied the effects of their respective inhibitors (i.e., cyclosporine A [CsA] and Z-LEHD-FMK, respectively) in primary cultures of chicken embryonic cecum epithelial cells using histopathological techniques, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assays, flow cytometry (FCM) and ELISA. Results indicated that the inhibitors significantly decreased (p < 0.01) DNA injury, apoptosis and caspase-9 and caspase-3 activity of chick embryo cecal epithelial cells at 24, 48, 72, 96 and 120 h after E. tenella infection. Thus, our data supported that mitochondria-dependent apoptotic pathways were involved in apoptosis of parasitised chick embryo cecal epithelial cells.

Effects of mannan oligosaccharide and virginiamycin on the cecal microbial community and intestinal morphology of chickens raised under suboptimal conditions.

There is an increasing movement against use of antibiotic growth promoters in animal feed. Prebiotic supplementation is a potential alternative to enhance the host's natural defense through modulation of gut microbiota. In the present study, the effect of mannan oligosaccharide (MOS) and virginiamycin (VIRG) on cecal microbial ecology and intestinal morphology of broiler chickens raised under suboptimal conditions was evaluated. MOS and VIRG induced different bacterial community structures, as revealed by denaturing gradient gel electrophoresis of 16S rDNA. The antibiotic treatment reduced cecal microbial diversity while the community equitability increased. A higher bacterial diversity was observed in the cecum of MOS-supplemented birds. Quantitative polymerase chain reaction results indicated that MOS changed the cecal microbiota in favor of the Firmicutes population but not the Bacteroidetes population. No difference was observed in total bacterial counts among treatments. MOS promoted the growth of Lactobacillus spp. and Bifidobacterium spp. in the cecum and increased villus height and goblet cell numbers in the ileum and jejunum. These results provide a deeper insight into the microbial ecological changes after supplementation of MOS prebiotic in poultry diets.

Differences in intestinal mucin dynamics between germ-free and conventionally reared chickens after mannan-oligosaccharide supplementation.

A germ-free (GF) chicken model was used to test 2 hypotheses: 1. microbial colonization of the gastrointestinal tract (GIT) influences mucin gene expression and mucin types; and 2. mannan oligosaccharide (MOS) supplementation affects GIT cells directly, without bacteria mediation, compared with bacterial-mediated effect (i.e., indirectly). Gnotobiotic isolators were used: 1) GF, 2) with a single bacteria population, and 3) conventionalized by exposure to cecal bacterial contents. Each was divided to 2 diet groups: with or without MOS (2 kg/t) for 1 wk. Results show that the absence of bacteria in the GIT caused a reduction in neutral and acidic goblet cell (GC) number and density, an increase in sulfated mucin, absence of sialylated GC, and reduced mucin 2 mRNA expression in the small intestine of GF compared with conventional birds. These results indicate a reduced development of mucin production and secretion in the absence of GIT bacteria implying a less mature small intestine mucosa, supporting our first hypothesis. Results from the single bacteria population group were not conclusive and did not support any of the hypotheses. Supplementation of MOS, regardless of microbial presence, caused a reduction in neutral GC number and density but increased neutral GC area. The MOS caused different effects on acidic mucins in conventional and GF birds, causing a reduction in sialylated GC number (conventional) and a reduction in sulfated GC density (GF), all supporting a direct effect of MOS in GF animals, in addition to an indirect effect via gut microflora.

Effect of whole wheat feeding on selected immune parameters in growing male turkeys.

The objective of this study was to determine the effect of whole wheat feeding on selected parameters of humoral and cell-mediated immunity in growing turkeys. A total of 210 one-day-old heavy-type Hybrid Converter male turkeys were randomly divided into three different dietary treatment groups, each consisting of 7 replicate pens of 10 birds per pen. Until 4 wk of age, all birds were fed a commercial diet formulated to meet nutrient requirements. From 5 to 12 wk of age, diets were composed of wheat (ground-pelleted or whole grain) and protein-fat-mineral-vitamin concentrate. The total wheat concentration in diets was 500 or 600 g/kg in the feeding periods of 5-8 and 9-12 wk of birds' age, respectively. Whole grain wheat had a 0, 50 or 100% share of the total wheat amount in the daily ration in treatment groups W0, W50 or W100, respectively. Commercial vaccines against ND (Newcastle disease) and TRT (Turkey rhinotracheitis) were administered to turkeys via the drinking water on days 20 and 30, respectively. Over the entire experiment, a significant linear decrease was observed in body weight gains (BWG) with increasing dietary levels of whole grain wheat. As a result the BWG of control turkeys (W0) were significantly higher than the BWG of group W100 birds (P = 0.002). A significant linear increase in feed conversion ratio (FCR) was observed with increasing dietary levels of whole grain wheat (P < 0.001). The levels of antibodies against TRT and ND viruses after immunization were significantly higher in both the W50 and W100 group, in comparison to group W0 (P = 0.006 and P = 0.001, respectively). Turkeys from group W50, in comparison to those from groups W0 and W100, had a significantly higher percentage of CD4+ T cell subpopulation within the lymphocytes isolated from blood and ileal mucosa, as well as CD4+ CD8+ and CD8+ T cell subpopulations within the blood immunocompetent cells (P = 0.022, P = 0.029, P = 0.009 and P = 0.011, respectively). In the cecal tonsils, the percentage of CD8+ T cell subpopulation was significantly lower in group W50 than in groups W0 and W100 (P = 0.014). The results of our study indicate that diluting diets with whole grain wheat stimulates the non-specific cell-mediated defense mechanisms of the gastrointestinal immune system in turkeys, thus positively affecting humoral response after vaccination.

Alternative luminal activation mechanisms for paneth cell α-defensins.

Paneth cell α-defensins mediate host defense and homeostasis at the intestinal mucosal surface. In mice, matrix metalloproteinase-7 (MMP7) converts inactive pro-α-defensins (proCrps) to bactericidal forms by proteolysis at specific proregion cleavage sites. MMP7(-/-) mice lack mature α-defensins in Paneth cells, accumulating unprocessed precursors for secretion. To test for activation of secreted pro-α-defensins by host and microbial proteinases in the absence of MMP7, we characterized colonic luminal α-defensins. Protein extracts of complete (organ plus luminal contents) ileum, cecum, and colon of MMP7-null and wild-type mice were analyzed by sequential gel permeation chromatography/acid-urea polyacrylamide gel analyses. Mature α-defensins were identified by N-terminal sequencing and mass spectrometry and characterized in bactericidal assays. Abundance of specific bacterial groups was measured by qPCR using group specific 16 S rDNA primers. Intact, native α-defensins, N-terminally truncated α-defensins, and α-defensin variants with novel N termini due to alternative processing were identified in MMP7(-/-) cecum and colon, and proteinases of host and microbial origin catalyzed proCrp4 activation in vitro. Although Paneth cell α-defensin deficiency is associated with ileal microbiota alterations, the cecal and colonic microbiota of MMP7(-/-) and wild-type mice were not significantly different. Thus, despite the absence of MMP7, mature α-defensins are abundant in MMP7(-/-) cecum and colon due to luminal proteolytic activation by alternative host and microbial proteinases. MMP7(-/-) mice only lack processed α-defensins in the small intestine, and the model is not appropriate for studying effects of α-defensin deficiency in cecal or colonic infection or disease.

Expression and cellular localization of monocarboxylate transporters (MCT2, MCT7, and MCT8) along the cattle gastrointestinal tract.

Fourteen members of the monocarboxylate transporter (MCT, SLC16) family have been identified, each having a different tissue distribution and substrate specificity. The expression of monocarboxylate transporters MCT1 and MCT4 have been studied in the gastrointestinal tract of ruminants; however, details of the expression of other MCT isoforms in the various parts of ruminant gastrointestinal tract are lacking. Reverse transcription with the polymerase chain reaction was used to study the regional distribution of MCT2, MCT3, and MCT5-MCT14 in the cattle gastrointestinal tract and verified the existence of MCT mRNA transcripts for MCT2, MCT3, MCT4, MCT7, MCT8, MCT9, MCT10, MCT13, and MCT14 in the ruminal and abomasal epithelia, mRNA transcripts for MCT2, MCT3, MCT4, MCT7, MCT8, MCT10, MCT13, and MCT14 in the jejunum, and mRNA transcripts for MCT2, MCT3, MCT4, MCT7, MCT8, MCT13, and MCT14 in the caecum of cattle. At the cellular level, immunohistochemical studies localized MCT2, MCT7, and MCT8 proteins in the cattle rumen, abomasum, jejunum, and caecum. This is the first study to detect the expression of various MCT isoforms in the gastrointestinal tract of a ruminant species. Our data suggest that these transporter proteins are involved in essential physiologic processes and are possible molecular targets for studying the regulation of the transport of short-chain monocarboxylates, aromatic amino acids, and thyroid hormones across the gastrointestinal tract of cattle.

Development of a feed additive to reduce caecal Campylobacter jejuni in broilers at slaughter age: from in vitro to in vivo, a proof of concept.

AIM: In vitro and in vivo challenge studies were undertaken to develop an in-feed additive of microencapsulated propionic, sorbic acids and pure botanicals to control Campylobacter jejuni in broilers at slaughter age. METHODS AND RESULTS: Organic acids (OA) and pure botanicals were tested in vitro against Camp. jejuni, whereas in vivo, chickens were fed either a control diet, or increasing doses of the additive for 42 days (experiment 1); in the second experiment, chickens received the additive at 0.1 or 0.3% from day 0 to 21 or from day 22 to 42. The additive consistently reduced Camp. jejuni caecal counts at any given dose (exp. 1) or inclusion plan (exp. 2). Moreover, it was able to reduce the number of goblet cells and modify mucin glycoconjugates biosynthesis pattern. CONCLUSIONS: We developed an additive that was effective in reducing Camp. jejuni in slaughter-age chickens even at low doses (0.1%). That efficacy was the result of the synergistic action between OA and botanicals. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides a strategy to reduce Camp. jejuni in broilers and, as a consequence, to improve the safety of the food chain. Moreover, data suggest that a treatment limited to the last weeks before slaughter would allow to save on inclusion of the additive throughout the whole production cycle.

Beta1 integrins are required for the invasion of the caecum and proximal hindgut by enteric neural crest cells.

Integrins are the major adhesive receptors for extracellular matrix and have various roles in development. To determine their role in cell migration, the gene encoding the beta1 integrin subunit (Itgb1) was conditionally deleted in mouse neural crest cells just after their emigration from the neural tube. We previously identified a major defect in gut colonisation by conditional Itgb1-null enteric neural crest cells (ENCCs) resulting from their impaired migratory abilities and enhanced aggregation properties. Here, we show that the migration defect occurs primarily during the invasion of the caecum, when Itgb1-null ENCCs stop their normal progression before invading the caecum and proximal hindgut by becoming abnormally aggregated. We found that the caecum and proximal hindgut express high levels of fibronectin and tenascin-C, two well-known ligands of integrins. In vitro, tenascin-C and fibronectin have opposite effects on ENCCs, with tenascin-C decreasing migration and adhesion and fibronectin strongly promoting them. Itgb1-null ENCCs exhibited an enhanced response to the inhibitory effect of tenascin-C, whereas they were insensitive to the stimulatory effect of fibronectin. These findings suggest that beta1 integrins are required to overcome the tenascin-C-mediated inhibition of migration within the caecum and proximal hindgut and to enhance fibronectin-dependent migration in these regions.

Immunomodulation in young laying hens by dietary folic acid and acute immune responses after challenge with Escherichia coli lipopolysaccharide.

We investigated the effects of dietary folic acid (FA) supplementation on immunological parameters in young laying hens under acute conditions of lipopolysaccharide (LPS) challenge. Twenty-four Shaver White laying hens at 24 wk were fed 2 diets in a completely randomized design. The diets were wheat-soybean based, with either 0 or 4 mg of supplemental FA per kilogram of diet. At 32 wk of age, 6 hens from each dietary treatment were injected intravenously with 8 mg/kg of BW of either LPS or saline. Four hours after injection, blood was collected and the hens were euthanized to obtain spleen and cecal tonsils. Heterophil:lymphocyte ratio, CD3+, CD4+, CD8+ T cells, and CD4+:CD8+ cells in the blood and spleen were not affected by dietary FA. Relative to saline-injected hens, LPS-injected hens had fewer (P < 0.05) CD3+, CD4+, CD8+, and CD4+:CD8+ cells in the blood, and no difference was found in the spleen. Total protein, albumin, and globulin were found to be higher (P < 0.05) in FA-supplemented hens compared with the control. However, total protein, albumin, and globulins decreased (P < 0.05) in the LPS-injected hens compared with the saline control. Expression of interleukin (IL)-1ß in cecal tonsils decreased (P < 0.05) in FA-supplemented hens, but no dietary influence was found on the expression of other genes in both the spleen and cecal tonsils. Lipopolysaccharide upregulated (P < 0.05) expression of IL-10 and interferon (IFN)-γ in the spleen, and IL-1ß, IL-10, and IFN-γ in the cecal tonsils, whereas the expression of Toll-like receptor (TLR)-4 and IL-8 was not influenced by LPS in the spleen and cecal tonsils. There was a diet × challenge interaction for total IgG, and cytokines IL-1ß and IL-18 in the spleen as well as IL-18 in the cecal tonsils. In conclusion, there were few interactions of dietary FA and LPS; however, FA increased biochemical constituents, enhanced generation of total IgG, as well as exhibiting pleoitropic effects in inflammatory responses.

Effect of recycled litter on immune cells in the cecal tonsils of chickens.

This experiment was conducted to study selected aspects of the gut immune response in broiler chicks reared on fresh or recycled litter that were fed diets with and without subtherapeutic antibiotic supplementation. All of the chicks were reared in pens that contained either fresh pine shavings (fresh litter) or litter that was recycled for 3 consecutive flocks (recycled litter). The experiment was a 2 × 2 factorial arrangement of treatments with 4 replicate pens (n = 4) per treatment. At 10 and 35 d of age, the cecal tonsils were analyzed for intestinal immune measurements. The cecal tonsils of birds reared on recycled litter had increased IL-1 mRNA (P < 0.01) and a lower percentage of CD4(+)CD25(+) cells at 10 and 35 d of age when compared with those of chicks reared on fresh litter. Birds fed diets supplemented with bacitracin had a reduction in CD4(+) cells (P = 0.01) at 10 d of age when compared with that of chicks that were not fed the antibiotic. The combination of bacitracin supplementation and fresh litter resulted in an approximate 10-fold increase in IL-10 mRNA (P = 0.01) at 10 d of age when compared with that of the unsupplemented chicks in fresh litter. Among those chicks that were not supplemented with bacitracin, the recycled-litter treatment resulted in 25-fold (P = 0.01) and 39-fold (P = 0.02) higher IL-4 mRNA levels at 10 and 35 d of age, respectively, when compared with those of the chicks reared on fresh litter. In conclusion, the intestinal immune response of birds reared on recycled litter is skewed toward an inflammatory response, whereas the fresh litter treatment was skewed toward an anti-inflammatory response. Bacitracin supplementation did not interact with the litter type to alter IL-1 mRNA levels in cecal tonsils, suggesting the low efficiency of bacitracin in alleviating the inflammatory response induced by recycled litter.

Pathobiology of Heterakis gallinarum mono-infection and co-infection with Histomonas meleagridis in layer chickens.

Little is known about the induction and modulation of gut-associated immune reactions after nematode infection in the chicken. The objective of the present study was to compare the pathogenesis, induction of immune reactions and electrophysiological changes of the gut after mono-infection with Heterakis gallinarum and after dual infection with H. gallinarum and Histomonas meleagridis in layer chickens. In two experiments 3-week-old chickens were inoculated with embryonated H. gallinarum eggs, which were positive for H. meleagridis. While birds of the first experiment were left untreated, those of the second experiment were treated with dimetridazol to prevent H. meleagridis co-infection. Mild to moderate histological lesions and local immune reactions with a significant increase in CD4(+), CD8α(+), TCRαß(+) and TCRδγ(+) cells in the lamina propria and induction of the T-helper type 2 (Th2) cytokine interleukin-13 dominated the H. gallinarum immune response at 2 weeks post infection. Co-infection with H. gallinarum and H. meleagridis induced an increase in mRNA expression of the T-helper type 1 (Th1) cytokine interferon-γ, a decrease in splenic CD4(+) cells and severe destruction of the caecal mucosa in association with strong T-cell infiltration in the caecal lamina propria. There was no obvious effect on the chloride secretion of the caecal epithelium, which was investigated once the mucosa had almost recovered from the infection, in either experiment. These results suggest that the local T-cell reactions to nematode infections in chickens may be comparable with mammals and may be shifted from a Th2-dominated to a Th1-dominated response when accompanied by a protozoan infection.

T-cell glucocorticoid receptor is required to suppress COX-2-mediated lethal immune activation.

Glucocorticoids, acting through the glucocorticoid receptor, potently modulate immune function and are a mainstay of therapy for treatment of inflammatory conditions, autoimmune diseases, leukemias and lymphomas. Moreover, removal of systemic glucocorticoids, by adrenalectomy in animal models or adrenal insufficiency in humans, has shown that endogenous glucocorticoid production is required for regulation of physiologic immune responses. These effects have been attributed to suppression of cytokines, although the crucial cellular and molecular targets remain unknown. In addition, considerable controversy remains as to whether glucocorticoids are required for thymocyte development. To assess the role of the glucocorticoid receptor in immune system development and function, we generated T-cell-specific glucocorticoid receptor knockout mice. Here we show that the T-cell is a critical cellular target of glucocorticoid receptor signaling, as immune activation in these mice resulted in significant mortality. This lethal activation is rescued by cyclooxygenase-2 (COX-2) inhibition but not steroid administration or cytokine neutralization. These studies indicate that glucocorticoid receptor suppression of COX-2 is crucial for curtailing lethal immune activation, and suggest new therapeutic approaches for regulation of T-cell-mediated inflammatory diseases.

Murine cecal patch M cells transport infectious prions in vivo.

We show that following oral inoculation, prions bind to ileal Peyer patch and cecal patch microfold cells (M cells) in vivo. Furthermore, we show evidence that the cecum acts a biological sump holding large concentrations of prions for relatively long periods, thus increasing the exposure time of cecal patch M cells. Our results show a critical initial step in the translocation of prions from the intestinal lumen of mammals in vivo, which is a precursor to infection.

Gut microbiota-derived short-chain fatty acids regulate IL-17 production by mouse and human intestinal γδ T cells.

Gut interleukin-17A (IL-17)-producing γδ T cells are tissue-resident cells that are involved in both host defense and regulation of intestinal inflammation. However, factors that regulate their functions are poorly understood. In this study, we find that the gut microbiota represses IL-17 production by cecal γδ T cells. Treatment with vancomycin, a Gram-positive bacterium-targeting antibiotic, leads to decreased production of short-chain fatty acids (SCFAs) by the gut microbiota. Our data reveal that these microbiota-derived metabolites, particularly propionate, reduce IL-17 and IL-22 production by intestinal γδ T cells. Propionate acts directly on γδ T cells to inhibit their production of IL-17 in a histone deacetylase-dependent manner. Moreover, the production of IL-17 by human IL-17-producing γδ T cells from patients with inflammatory bowel disease (IBD) is regulated by propionate. These data contribute to a better understanding of the mechanisms regulating gut γδ T cell functions and offer therapeutic perspectives of these cells.

Segregation of Na/H exchanger-3 and Cl/HCO3 exchanger SLC26A3 (DRA) in rodent cecum and colon.

The colon is believed to absorb NaCl via the coupled operation of apical Na/H exchanger-3 (NHE3) and Cl/HCO(3) exchanger SLC26A3 (DRA). Efficient coupling requires that NHE3 and DRA operate in close proximity within common luminal and cytosolic microenvironments. Thus we examined whether these proteins coexist along the apical margin of surface enterocytes by quantitative immunofluorescence microscopy in consecutive colon segments from nonfasted mice and rats. The cecocolonic profiles of NHE3 and DRA expression were roughly inverse; NHE3 was highest in proximal colon (PC) and negligible in distal colon, whereas DRA was absent in early PC and highest in the late midcolon, and DRA was prominent in the cecum whereas NHE3 was not. NHE3 and DRA coexisted only in the middle third of the colon. The consequences of unpaired NHE3/DRA expression on mucosal surface (subscript MS) pH and Na(+) concentration ([Na(+)]) were assessed in nonfasted rats in situ using miniature electrodes. In the cecum, where only DRA is expressed, pH(MS) was approximately 7.5, markedly higher than underlaying stool (6.3), consistent with net HCO(3)(-) secretion. In the early PC, where NHE3 is not expressed with DRA, pH(MS) was acidic (6.2), consistent with unopposed H(+) secretion. [Na(+)](MS) was approximately 60 mM in the cecum, decreased along the PC to approximately 20 mM, and declined further to approximately 10 mM distally. Cl(-) was secreted into the PC, then reabsorbed distally. Our results suggest a model in which 1) unpaired DRA activity in the cecum maintains an alkaline mucosal surface that could neutralize fermentative H(+); 2) unpaired NHE3 activity in the early PC preserves an acidic mucosal surface that could energize short-chain fatty acid absorption; and 3) coupled NHE3/DRA activities in the midcolon allow for vigorous NaCl absorption at a neutral pH(MS).

Morphological characterization of Cryptosporidium parvum life-cycle stages in an in vitro model system.

Cryptosporidium parvum is a zoonotic protozoan parasite that mainly affects the ileum of humans and livestock, with the potential to cause severe enteric disease. We describe the complete life cycle of C. parvum in an in vitro system. Infected cultures of the human ileocecal epithelial cell line (HCT-8) were observed over time using electron microscopy. Additional data are presented on the morphology, development and behavioural characteristics of the different life-cycle stages as well as determining their time of occurrence after inoculation. Numerous stages of C. parvum and their behaviour have been visualized and morphologically characterized for the first time using scanning electron microscopy. Further, parasite-host interactions and the effect of C. parvum on host cells were also visualized. An improved understanding of the parasite's biology, proliferation and interactions with host cells will aid in the development of treatments for the disease.

Large intestinal mast cell count and proteinase expression is associated with larval burden in cyathostomin-infected horses.

REASONS FOR PERFORMING STUDY: Cyathostomins are the principal pathogenic nematode of equidae worldwide. In other species mast cell (MC) proteinases, in particular chymases, appear to have protective roles. Knowledge of the equine intestinal immune response to cyathostomins is limited. OBJECTIVE: To investigate MC numbers and proteinase expression in equine cyathostomin-infected large intestine. HYPOTHESIS: MC populations in the large intestine are positively associated with cyathostomin burden and predominantly express chymase. METHODS: The caecal cyathostomin burden of naturally infected horses (n = 25) was determined by luminal counts and pepsin digest (mural count). MC were identified and enumerated in caecal tissue using toluidine blue (TB). Immunofluorescent labelling with polyclonal rabbit antibodies was used to demonstrate expression of equine tryptase and the chymase equine mast cell proteinase-1 (eqMCP-1) in Carnoy's fixed caecal sections. RESULTS: Significant positive linear relationships were found between TB-stained mucosal and submucosal MC counts and total cyathostomin burden (P<0.001, r² >36%), and both luminal (P<0.010, r² >25%) and mural (P<0.001, r² >36%) larval counts. Similar relationships were found with mucosal and submucosal chymase and tryptase-labelled MC counts (total: P<0.004, r² >29%; luminal: P<0.004, r² >30%; and mural: P<0.030, r² >19%). With all three MC labels, mean MC counts were higher in the submucosa compared to the mucosa (P<0.001). All caecal MC appeared to express chymase, with a small number of MC expressing both tryptase and chymase. CONCLUSIONS AND POTENTIAL RELEVANCE: Large intestinal MC counts are significantly associated with cyathostomin burden, with a predominance of chymase-positive MC. The burden is significantly associated with expression of MC proteinases, supporting their likely involvement in the intestinal immune response to cyathostomin infection. Further work to investigate the kinetics of proteinase expression, the possibility of differential proteinase expression and the role of these MC proteinases is warranted.

Chicken chemokine receptors in T cells isolated from lymphoid organs and in splenocytes cultured with concanavalin A.

Chemokine receptors guide immune cells to specific organs during health and disease. The mRNA content of the chemokine receptors CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CXCR4, CXCR5, and CX3CR1 in CD4(+) cells (T-helper cells) isolated from blood, bursa, cecal tonsil, spleen, and thymus and in CD8(+) cells (T-cytotoxic cells) isolated from blood, cecal tonsil, spleen, and thymus were investigated. The CD4(+) cells isolated from thymus had the highest amount of CCR7 and CCR8 mRNA. The CD4(+) cells isolated from bursa, cecal tonsil, and thymus had the highest amount of CCR5 mRNA. The CD4(+) cells isolated from cecal tonsils had the highest amount of CCR9 mRNA. The CD4(+) cells isolated from bursa and thymus had the highest amount of CXCR5 mRNA. The CD8(+) cells isolated from cecal tonsil had the highest mRNA amount of all receptors studied except CCR9 and CX3CR1. The CD4(+) cells treated with concanavalin A had increased CCR2, CCR4, CCR7, CCR8, and CXCR5 mRNA amounts at 24 h of stimulation. The CD8(+) cells treated with concanavalin A had increased CCR4 mRNA at 72 h, increased CCR6 mRNA at 24 h, and decreased CCR8 and CXCR4 mRNA at 24 h of stimulation.

Intravital imaging of eosinophils: Unwrapping the enigma.

Eosinophils are traditionally associated with allergic and parasitic inflammation. More recently, eosinophils have also been shown to have roles in diverse processes including development, intestinal health, thymic selection, and B-cell survival with the majority of these insights being derived from murine models and in vitro assays. Despite this, tools to measure the dynamic activity of eosinophils in situ have been lacking. Intravital microscopy is a powerful tool that enables direct visualization of leukocytes and their dynamic behavior in real-time in a wide range of processes in both health and disease. Until recently eosinophil researchers have not been able to take full advantage of this technology due to a lack of tools such as genetically encoded reporter mice. This mini-review examines the history of intravital microscopy with a focus on eosinophils. The development and use of eosinophil-specific Cre (EoCre) mice to create GFP and tdTomato fluorescent reporter animals is also described. Genetically encoded eosinophil reporter mice combined with intravital microscopy provide a powerful tool to add to the toolbox of technologies that will help us unravel the mysteries still surrounding this cell.